Chronic Kidney Disease (CKD) affects over 19M people in the United States and is frequently a consequence of metabolic disorders involving obesity, diabetes, and hypertension. Examination of the data reveals that the rate of increase is due to the development of renal failure secondary to hypertension and non-insulin dependent diabetes mellitus (NIDDM) (United States Renal Data System: Costs of CKD and ESRD. ed. Bethesda, Md., National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, 2007 pp 223-238)—two diseases that are also on the rise worldwide. Obesity, hypertension, and poor glycemic control have all been shown to be independent risk factors for kidney damage, causing glomerular and tubular lesions and leading to proteinuria and other systemically-detectable alterations in renal filtration function (Aboushwareb, et al., World J Urol, 26: 295-300, 2008; Amann, K. et al., Nephrol Dial Transplant, 13: 1958-66, 1998). CKD patients in stages 1-3 of progression are managed by lifestyle changes and pharmacological interventions aimed at controlling the underlying disease state(s), while patients in stages 4-5 are managed by dialysis and a drug regimen that typically includes anti-hypertensive agents, erythropoiesis stimulating agents (ESAs), iron and vitamin D supplementation. According to the United States Renal Data Service (USRDS), the average end-stage renal disease (ESRD) patient expends >$600 per month on injectable erythropoiesis-stimulating agents (ESAs), Vitamin D supplements, and iron supplements (United States Renal Data System: Costs of CKD and ESRD. ed. Bethesda, Md., National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, 2007 pp 223-238). When paired with the annual average cost of dialysis ($65,405), the healthcare cost for maintenance of a single patient rises to >$72,000/yr (United States Renal Data System: Costs of CKD and ESRD. ed. Bethesda, Md., National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, 2007 pp 223-238)—a number that reflects only standard procedural costs and does not include treatment of other complications, emergency procedures, or ancillary procedures such as the placement of vascular grafts for dialysis access. Combined medicare costs for CKD and ESRD in 2005 totaled $62B—representing 19% of all medicare spending for that year (United States Renal Data System: Costs of CKD and ESRD. ed. Bethesda, Md., National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, 2007 pp 223-238). Kidney transplantation is an effective option for stage 4-5 patients as a pre-emptive measure to avoid dialysis or when dialysis is no longer sufficient to manage the disease state, but the number of stage 5 CKD patients in the US (>400,000) who could benefit from whole kidney transplant far exceeds the number of suitable donor kidneys available in any given year (˜16,000) (Powe, N R et al., Am J Kidney Dis, 53: S37-45, 2009). Thus, new treatment paradigms are needed to delay or reduce dependency on dialysis and to fill the void left by the shortage of donor kidneys.
Progressive renal disease results from a combination of the initial disease injury (e.g, hypertension), followed by a maladaptive renal response to that injury. Such a response includes the production of pro-inflammatory and pro-fibrotic cytokines and growth factors. Therefore, one strategy to slow CKD progression is to ameliorate the inflammatory and fibrotic response as well as mitigate or reverse renal degeneration through the repair and/or regeneration of renal tissue.
Chronic renal failure is prevalent in humans as well as some domesticated animals. Patients with renal failure experience not only the loss of kidney function (uremia), but also develop anemia due to the inability of the bone marrow to produce a sufficient number of red blood cells (RBCs) via erythropoiesis. Erythroid homeostasis is dependent on both the production of erythropoietin (EPO) by specialized interstitial fibroblasts that reside in the kidney and the ability of targeted erythroid progenitors in the bone marrow to respond to EPO and manufacture more RBCs. The anemia of renal failure is due to both reduced production of EPO in the kidney and the negative effects of uremic factors on the actions of EPO in the bone marrow.
To date, clinical approaches to the treatment of chronic renal failure involve dialysis and kidney transplantation for restoration of renal filtration and urine production, and the systemic delivery of recombinant EPO or EPO analogs to restore erythroid mass. Dialysis offers survival benefit to patients in mid-to-late stage renal failure, but causes significant quality-of-life issues. Kidney transplant is a highly desired (and often the only) option for patients in the later stages of renal failure, but the supply of high-quality donor kidneys does not meet the demand for the renal failure population. Bolus dosing with recombinant EPO to treat anemia has now been associated with serious downstream health risks, leading to black box warnings from the FDA for the drug, and necessitating further investigation into alternative treatments to restore erythroid homeostasis in this population. Preclinical investigations have examined in vivo efficacy and safety of EPO-producing cells that have been generated via gene therapy approaches. These studies have shown that it is possible to transiently stimulate erythropoiesis and RBC number by in vivo delivery of epo-producing cells. However, to date, none of these approaches have offered regulated erythroid homeostasis or long-term in vivo functionality. Consequently, HCT and RBC number are often increased beyond normal values, leading to polycythemia vera and other complications. Delivery of EPO-producing cells that are therapeutically-relevant and provide advantages over delivery of recombinant EPO must not only increase HCT, but should restore erythroid homeostasis, with both positive and negative regulatory mechanisms intact. It is important to note that EPO-deficient anemias, while prevalent in patients with kidney disease, can also develop as a result of other disease states, including heart failure, multi-organ system failure, and other chronic diseases.
The kidney is a unique organ comprised of many different specialized cell types (>10), all of which originate developmentally from the intermediate mesoderm but at maturity form morphologically and functionally distinct compartments, and anatomical units that act in concert to provide filtration of the blood, production of urine, regulation of acid-base and electrolyte balance, and regulated endocrine functions such as the production of erythropoietin (Epo), Vitamin D, renin, and angiotensin. The cellular compartments of the kidney are heavily interdependent for homeostatic function, as highlighted by the following examples. Cells of the afferent arterioles act in concert with specialized tubular cells in the thick ascending limb of the loop of Henle (Macula Densa) to regulate blood flow through the glomerulus (Castrop, H. Acta Physiol (Oxf), 189: 3-14, 2007). Protein handling by the kidney is orchestrated by the fenestrated endothelial cells, podocytes, and basement membrane of the glomerulus paired with the receptor-mediated endocytosis and resorption of protein from the glomerular filtrate by specialized proximal tubular cells (Jarad, G & Miner, J H. Curr Opin Nephrol Hypertens, 18: 226-32, 2009). Production of active vitamin D by tubular cells regulates homeostasis of interstitial cells through direct and indirect mechanisms that control extracellular matrix deposition, conversion of interstitial cells to myofibroblasts, and epithelial-mesenchymal transformation (Tan, X, et al. J Steroid Biochem Mol Biol, 103: 491-6, 2007). Regardless of the specific example, all cell-cell interactions in the kidney are at least partially dependent on spatial and architectural relationships. At the cellular level, progression of CKD may involve loss of a particular cell type or loss of function of one or more cell types due to cellular insufficiencies or loss of homeostatic cell-cell interactions. Thus, successful regenerative approaches to the treatment of CKD must re-establish homeostasis in part through restoration of cellular organization and intercellular communication.
Augmentation of specific kidney functions, such as tubular transport or production of Epo, has been contemplated with the intention of reducing the morbidity and mortality associated with progression of CKD. The majority of cell-based treatment approaches for kidney disease have focused on therapeutic intervention of acute renal failure (ARF) with stem or progenitor cell types (Hopkins, C, et al. J Pathol, 217: 265-81, 2009). There have been many preclinical studies involving the delivery of various cell types immediately before or after induction of ARF, including intrarenal or systemic delivery of MSCs (Humphreys B D & Bonventre J V, Annu Rev Med 2008, 59:311-325), endothelial progenitors (EPCs) (Chade A R, et al., Circulation 2009, 119:547-557, Patschan D, et al., Curr Opin Pharmacol 2006, 6:176-183), and fetal cells or tissue rudiments (Hammerman M R, Curr Opin Nephrol Hypertens 2001, 10:13-17; Kim S S, et al, Stem Cells 2007, 25:1393-1401; Marshall D, et al., Exp Physiol 2007, 92:263-271; Yokoo T, et al., J Am Soc Nephrol 2006, 17:1026-1034). An extracorporeal hollow-fiber filtration device containing renal tubular cells was tested as an adjunct to traditional dialysis for the treatment of ARF in humans (Ding, F & Humes, H D. Nephron Exp Nephrol, 109: e118-22, 2008, Humes, H D, et al. Kidney Int, 66: 1578-88, 2004, Humes, H D, et al. Nat Biotechnol, 17: 451-5, 1999). Transplantation of mesenchymal stem cells via the renal artery is also being tested clinically in a population of patients at high risk for an ARF episode secondary to cardiovascular surgical procedures (Westenfelder, C. Experimental Biology. New Orleans, L A, 2009). Limited preclinical studies have been conducted that address cell-based therapeutic intervention of CKD (Chade, A R, et al. Circulation, 119: 547-57, 2009, Eliopoulos, N, et al. J Am Soc Nephrol, 17: 1576-84, 2006, Kucic, T, et al. Am J Physiol Renal Physiol, 295: F488-96, 2008). The combination of fetal kidney rudiments+/−mesenchymal stem cells has been investigated in rodents (Yokoo, T, et al. Transplantation, 85: 1654-8, 2008, Yokoo, T, et al. J Am Soc Nephrol, 17: 1026-34, 2006), where it is clear that whole fetal kidney tissue transplanted to an appropriate environment, such as the omentum, can develop into kidney structures with limited function. However, the therapeutic role of the MSCs as a component of the fetal tissue rudiment is unclear, and sourcing of human fetal kidney tissue for therapeutic purposes poses many operational and ethical challenges. In other studies, cells derived from healthy donor bone marrow were transplanted into irradiated COL4A3 (−/−) mice, a model of Alport Syndrome with glomerulonephritis, protein loss, and fibrosis, where they partially slowed progression in the model via replacement of leaky glomerular podocytes with healthy cells lacking the collagen gene mutations (Prodromidi, E I, et al. Stem Cells, 24: 2448-55, 2006, Sugimoto, H, et al. Proc Natl Acad Sci USA, 103: 7321-6, 2006). Cell transplantation was credited with stabilization of sCREAT, BUN, and sodium levels, but untreated/kidney-damaged controls were not presented for comparison in the studies 24. Chade et al employed a swine model of unilateral renal artery stenosis to examine the effects of autologous EPCs, delivered intrarenally 6 weeks post-injury (Chade A R, et al., Circulation 2009, 119:547-557). The EPCs improved tubulo-interstitial fibrosis somewhat, significantly improved glomerulosclerosis, and improved renal blood flow, although no change in blood pressure was observed with treatment (Chade A R, et al., Circulation 2009, 119:547-557). To date, studies that examined the in vivo efficacy of cell-based therapies for CKD have yielded transient and/or partial effects, and few studies have collected both systemic and histologic evidence of function. The limited number of studies that provide evidence of clinically-relevant benefits after intervention in progressive models of CKD raises questions about the potential of cell-based therapies to restore renal function completely. However, regenerative therapies that stabilize renal function and delay progression can address an unmet medical need within this patient population.
Reproducible in vivo model(s) of progressive CKD are essential for assessment of the therapeutic potential of candidate treatments. While models of ARF are numerous and include a variety of chemical- or ischemia/reperfusion-induced tubular injuries, there are fewer models of CKD that are progressive and terminal without significant intervention. The two-step 5/6 nephrectomy procedure in rats reproducibly generates a terminal and progressive state of renal failure, resulting in systemically- and histologically-detectable disease complete with several key features of CKD, including hypertension, reduced glomerular filtration rate (GFR), elevated serum creatinine (sCREAT) and BUN, glomerular and tubulo-interstitial fibrosis, hyperlipidemia, hyperphosphatemia, and anemia (Kaufman, J M, et al. Kidney Int, 6: 10-7, 197422, Platt, R, et al. Clin Sci (Lond), 11: 217-31, 1952, Ormrod, D & Miller, T. Nephron, 26: 249-54, 1980, Brenner, B M. Am J Physiol, 249: F324-37, 1985). The presence of these clinically-relevant features, combined with technical reproducibility and commercial availability provided the basis for selection of this model for the studies described herein.
Thus, new treatment paradigms are needed that provide substantial and durable augmentation of kidney functions, to slow progression and improve quality of life in this patient population and reduce the annual cost burden on the healthcare system. Regenerative medicine technologies may provide next-generation therapeutic options for CKD.